The long term goal of the proposed technology development is to take the current applications of the protein kinase (PK) substrate chips to the level suitable for proteome-scale applications and for developing clinical diagnostics. Recent years have witnessed dramatic expansion in our understanding of PK biology. In addition to the manifested intricate networks of the interactions often simultaneously or sequentially involving multiple PK enzymes which may target more than one protein, PK associations with human diseases are well-documented and more than 200 PKs are linked to human disease loci. To be able to effectively address the questions associated with these complex systems, multiplexing assay/screening technologies based on specific PK substrates have become increasingly important. Currently, only a limited number of PK assay choices are available and these are mostly low throughput or only for qualitatively assessment of substrate-kinase activities and specificities; there is no supporting platform for the proteome-scale multiplexing profiling and mapping of specific PK substrates in tens of thousands of sequences. Therefore, the development of a robust, quantitative, comprehensive, and high throughput PK assay platform is a significant step for addressing the rising needs, and moreover, a quantitative approach for enzymatic reaction measurements on peptide microchips will be promising for enabling clinical diagnosis applications. We propose the development of novel microchips of PK substrates and methods to fulfill these requirements. We have a collaboration team with expertise in microfabrication of bioassay devices, parallel synthesis, peptide chemistry, cell signaling, and cell biology ensuring useful assays to be developed and the in vitro microchip experimental results verified in in vivo systems. At the completion of the project (phases I and II), the catalog and custom PK substrate peptide microchips for quantitative, parallel measurements of the activities of a list of kinases against proteome-scale substrate sequences will be close to the market for research use. The established methods of on-chip assays will be used for high throughput PK specificity mapping, PK profiling, novel PK identification, PK inhibitor screening, and the development of diagnostic applications. [unreadable] [unreadable]